Comparison of fluticasone propionate and budesonide on COPD macrophage and neutrophil function

Figures & data

Table 1 Demographics of COPD subjects

Demographics	COPD patients (n=29)
Sex, male:female (n)	15:14
Age (years)	69±1.4
Smoking history (pack-years)	28.5±4.3
FEV1 (L)	1.3±0.1
FEV1 (% predicted)	56.8±2.7
FVC (L)	2.7±0.2
FVC (% predicted)	84.5±4.7
FEV1:FVC ratio	0.50±0.03

Note: Data presented as mean ± SEM, except where indicated.

Figure 1 Effects of 1 hour’s pretreatment with fluticasone propionate or budesonide on phagocytosis of beads or bacteria at 1 hour and 4 hours.

Notes: Monocyte-derived macrophages from COPD patients (n=20–24) were untreated (UC [untreated control]) or pretreated for 1 hour with fluticasone propionate (○), budesonide (●), or drug vehicle (V) prior to incubation with either fluorescently labeled beads (A, D), H. influenzae (B, E) or S. pneumoniae (C, F) for 1 hour (A–C) or 4 hours (D–F). Phagocytosis was measured by fluorimetry and reported as relative fluorescent units (RFUs). Data are mean ± SEM; *P<0.05 for differences between budesonide and vehicle (V).

Figure 2 Effects of 18 hours’ pretreatment with fluticasone propionate or budesonide on phagocytosis of beads or bacteria at 1 hour and 4 hours.

Notes: Monocyte-derived macrophages from COPD patients (n=22) were untreated (UC [untreated control]) or pretreated for 18 hours with fluticasone propionate (○) or budesonide (●) at indicated concentrations or drug vehicle (V) prior to incubation with fluorescently labeled beads (A, D), H. influenzae (B, E) or S. pneumoniae (C, F) for 1 hour (A–C) or 4 hours (D–F). Phagocytosis was measured by fluorimetry and reported as relative fluorescent units (RFUs). Data are mean ± SEM; ^#P<0.05, ^##P<0.01 between budesonide and fluticasone propionate.

Figure 3 Effects of 1 hour’s pretreatment with fluticasone propionate or budesonide on release of CXCL8, IL6, and TNFα induced by phagocytosis of beads or bacteria at 4 hours.

Notes: (A–C) Monocyte-derived macrophages from COPD patients (n=12) were left not treated (NT), or pretreated for 1 hour with drug vehicle (V) prior to incubation with fluorescently labeled beads, Haemophilus influenzae (HI), or Streptococcus pneumoniae (SP) bacteria for 4 hours. Supernatants were analyzed by ELISA for CXCL8 (A), IL6 (B), and TNFα (C) release. Data are mean ± SEM; **P<0.01, ***P<0.001 between no treated (NT) and treatment. (D–L) Monocyte-derived macrophages from COPD patients (n=12) were pretreated for 1 hour with fluticasone propionate (○) or budesonide (●) at indicated concentrations or drug vehicle (V) prior to incubation with fluorescently labeled beads or bacteria for 4 hours. Supernatants were analyzed by ELISA for CXCL8, IL6, and TNFα release. Data are percentage responses compared to vehicle control, expressed as mean ± SEM; *P<0.05, **P<0.01 between drugs and vehicle (V).

Figure 4 Effects of 18 hour's pretreatment with fluticasone propionate or budesonide on release of CXCL8, IL6, and TNFα induced by phagocytosis of beads or bacteria at 4 hours.

Notes: Monocyte-derived macrophages from COPD patients (n=12) were pretreated for 18 hours with fluticasone propionate (○) or budesonide (●) at indicated concentrations or drug vehicle (V) prior to incubation with fluorescently labeled beads (A–C), H. influenzae (D–F), or S. pneumoniae (G–I) or bacteria for 4 hours. Supernatants were analyzed by ELISA for CXCL8, IL6, or TNFα release. Data are percentage responses compared to vehicle control, expressed as mean ± SEM, with no statistical differences observed.

Table 2 Effects of 1 hour’s pretreatment with fluticasone propionate or budesonide on scavenger-receptor expression

Receptor	NT	HI	HI + V	HI + FP	HI + Bud	SP	SP + V	SP + FP	SP + Bud
Percentage of cells expressing receptors
CD36	42 (12)	55 (9)	55 (8)	64 (8)	53 (9)	50 (10)	45 (11)	38 (9)	42 (10)
CD206	47 (13)	68 (8)	72 (6)	71 (8)	66 (10)	56 (12)	59 (9)	53 (9)	52 (10)
CD163	93 (2)	92 (6)	99 (1)	99 (1)	96 (2)	94 (1)	98 (1)	98 (1)	96 (2)
TLR2	4 (1)	21 (5)**	24 (4)	25 (3)	17 (5)	11 (3)	6 (2)	5 (1)	5 (2)
TLR4	3 (1)	22 (6)**	20 (4)	18 (4)	13 (5)	12 (4)	5 (1)	4 (1)	4 (2)
MARCO	24 (12)	45 (18)	41 (14)	33 (15)	33 (16)	61 (10)	69 (10)	62 (12)	59 (13)
Median fluorescence intensity
CD36	5.6 (0.8)	5.7 (0.7)	6.3 (1.0)	5.8 (0.7)	5.7 (0.7)	5.8 (0.8)	5.2 (0.6)	4.7 (0.5)	5.0 (0.6)
CD206	2.6 (0.4)	2.8 (0.5)	2.6 (0.4)	2.7 (0.4)	2.5 (0.3)	2.7 (0.5)	2.8 (0.6)	2.3 (0.3)	2.4 (0.3)
CD163	1.5 (0.4)	2.0 (0.6)	2.3 (0.6)	3.1 (1.1)	2.4 (0.8)	1.8 (0.4)	1.8 (0.5)	1.9 (0.6)	1.6 (0.4)
TLR2	2.1 (0.3)	2.3 (0.3)	2.3 (0.3)	2.3 (0.3)	2.2 (0.3)	2.0 (0.2)	1.9 (0.2)	2.0 (0.2)	2.0 (0.2)
TLR4	6.1 (0.7)	6.5 (0.9)	6.5 (0.9)	6.6 (0.9)	6.5 (0.8)	5.9 (0.8)	6.0 (0.8)	5.9 (0.7)	5.5 (0.8)
MARCO	9 (2)	10 (2)	10 (2)	11 (3)	9 (2)	10 (1)	11 (2)	10 (2)	10 (2)

Notes: Monocyte-derived macrophages from COPD patients (n=6–8) were pretreated for 1 hour with cell media only (not-treated [NT], Haemophilus influenzae [HI], and Streptococcus pneumoniae [SP] groups), drug vehicle (V), 10⁻⁵ M fluticasone propionate (FP), or 10⁻⁵ M budesonide (Bud), prior to incubation with fluorescently labeled HI or SP bacteria for 4 hours. Cells were labeled with antibodies against indicated scavenger receptors and receptor expression analyzed by flow cytometry. Data are presented as mean (SEM).

** P<0.01 compared to NT.

Figure 5 Effects of fluticasone propionate or budesonide on intracellular killing of Haemophilus influenzae or Streptococcus pneumoniae by MDMs from COPD patients.

Notes: MDMs from COPD patients (n=7) were untreated (UC [untreated control]) or pretreated with fluticasone propionate (○) or budesonide (●) at indicated concentrations or drug vehicle (V), for 1 hour, and subsequently MDMs were incubated with H. influenzae (A) or S. pneumoniae (B) for 4 hours, antibiotics added to kill extracellular bacteria, MDMs lysed, and cell lysates plated on agar plates. After 12 hours, bacteria colonies were counted and colony-forming units (CFUs) calculated. Data presented as mean ± SEM, with no statistical differences observed; n=10 for H. influenzae; n=7 for S. pneumoniae.
Abbreviation: MDMs, monocyte-derived macrophages.

Figure S1 Effects of fluticasone propionate or budesonide on viability of MDMs after phagocytosis.

Notes: MDMs from COPD patients (n=20–24) were untreated (UC [untreated control]), or pretreated for 1 hour (A–F) or 18 hours (G–L) with fluticasone propionate (○), budesonide (●), or drug vehicle (V) prior to incubation with fluorescently labeled beads or bacteria for 1 hour or 4 hours. Cell viability was measured by MTT assay. Data presented as percentage viability compared to UC and shown as mean ± SEM; *P<0.05 between UC and drug.

Abbreviation: MDMs, monocyte-derived macrophages.

Figure S2 Flow cytometry gating strategy.

Notes: (A–H) Monocyte-derived macrophages were gated by the forward-scatter (FSc) vs side-scatter (SSc) population. Negative expression was gated on untreated population (red) or secondary antibody (MARCO), and a shift to the right indicates positive expression of receptors. Graphs show untreated cells (blue), H. influenzae-positive cells (light green), and cells treated with fluticasone propionate (orange), budesonide (dark green), and vehicle control (pink).

Figure S3 Effects of fluticasone propionate or budesonide on percent phagocytosis of Haemophilus influenzae and Streptococcus pneumoniae by COPD neutrophils.

Notes: Neutrophils from COPD patients were pretreated with fluticasone propionate (○) or budesonide (●) at indicated concentrations or drug vehicle (V) for 1 hour and subsequently incubated with H. influenzae (A–D) or S. pneumoniae (E–H) for 5, 10, 15, or 60 minutes, cells washed, fixed in 4% paraformaldehyde, and fluorescence measured by flow cytometry. Graphs show percentage of neutrophils that phagocytosed bacteria. Data shown as mean ± SEM with no statistical differences observed; n=7.

Figure S4 Effects of fluticasone propionate or budesonide on amount of Haemophilus influenzae and Streptococcus pneumoniae phagocytosed by COPD neutrophils.

Notes: Neutrophils from COPD patients were pretreated with fluticasone propionate (○) or budesonide (●) at indicated concentrations or drug vehicle (V) for 1 hour and subsequently incubated with H. influenzae (A–D) or S. pneumoniae (E–H) for 5, 10, 15, or 60 minutes, cells washed, fixed in 4% paraformaldehyde, and fluorescence measured by flow cytometry. Graphs show the amount of phagocytosed bacteria expressed as median fluorescence intensity (MFI). Data shown as mean ± SEM with no statistical differences observed; n=7.

Table S1 Effects of 18 hours’ pretreatment with fluticasone propionate (FP) or budesonide (Bud) on scavenger-receptor expression

Receptor	NT	HI	HI + V	HI + FP	HI + Bud	SP	SP + V	SP + FP	SP + Bud
Percentage of cells expressing receptors
CD36	41 (13)	50 (8)	59 (8)	60 (7)	49 (8)	41 (19)	41 (9)	35 (8)	39 (8)
CD206	48 (12)	65 (8)	68 (7)	70 (6)	63 (8)	50 (11)	61 (8)	55 (8)	60 (10)
CD163	96 (3)	97 (2)	96 (3)	97 (2)	97 (1)	96 (2)	96 (3)	97 (2)	97 (2)
TLR2	4 (2)	21 (3)**	28 (5)	27 (6)	26 (4)	6 (2)	7 (2)	9 (4)	9 (4)
TLR4	2 (1)	20 (4)**	23 (4)	23 (7)	22 (5)	8 (3)	7 (3)	7 (4)	9 (5)
MARCO	17 (16)	39 (16)	35 (7)	35 (16)	37 (19)	58 (11)	53 (14)	45 (14)	50 (13)
Median fluorescence intensity
CD36	5.3 (0.6)	5.3 (0.6)	5.9 (0.7)	5.9 (0.7)	5.3 (0.7)	4.9 (0.6)	5.0 (0.6)	4.8 (0.5)	4.5 (0.5)
CD206	2.1 (0.2)	2.5 (0.5)	2.4 (0.3)	2.4 (0.3)	2.0 (0.3)	2.0 (0.3)	2.0 (0.2)	2.0 (0.2)	2.0 (0.3)
CD163	1.6 (0.3)	1.9 (0.5)	2.4 (0.8)	2.9 (1.0)	2.7 (0.8)	1.6 (0.4)	1.8 (0.5)	2.8 (0.9)	2.4 (0.7)
TLR2	2.0 (0.2)	2.4 (0.3)	2.4 (0.3)	2.4 (0.3)	2.4 (0.3)	2.0 (0.2)	2.0 (0.2)	2.0 (0.2)	2.3 (0.3)
TLR4	6.3 (0.7)	6.6 (0.9)	6.6 (0.9)	6.7 (1.0)	6.8 (0.9)	6.0 (0.9)	6.0 (0.8)	6.5 (0.9)	6.5 (0.9)
MARCO	9 (2.0)	10 (2.0)	10 (2.0)	10 (2.0)	10 (2.0)	10 (1.0)	10 (1.0)	10 (2.0)	10 (2.0)

Notes: Monocyte-derived macrophages from COPD patients (n=6–8) were pretreated for 18 hours with cell media only (not-treated [NT], Haemophilus influenzae [HI], and Streptococcus pneumoniae [SP] groups), drug vehicle (V), 10⁻⁵ M FP or 10⁻⁵ M Bud, prior to incubation with fluorescently labeled HI or SP bacteria for 4 hours. Cells were labeled with antibodies against scavenger receptors indicated and receptor expression analyzed by flow cytometry. Data expressed as mean (SEM);

** P<0.01 compared to NT.